T-cells mediate the cellular immune response by attacking virally infected cells, cancer cells and parasites. Stimulation of T-cell receptors initiates a signal transduction cascade that results in the release of numerous proteins called cytokines, such as interleukin-2. Extracellular signal-regulated kinases (ERK), MEK (for MAPK or ERK kinase), and phosphatase of activated cells (PAC1)are all known to be expressed in the T-cells. The main goals of this proposal are to elucidate the mechanism(s) of MEK and ERK regulation in T-cells and to dissect the involvement of the two isoforms of MEK (MEK1) and MEK2)in T- cell signal transduction. The experiments presented will investigate the role of MEK in T-cell signal transduction and determine the effect of feedback phosphorylation of MEK by activated ERK. This will be accomplished by overexpressing either wild type MEK or MEK mutants by transient transfection in T-cells and examining the effects of mitogen stimulation on MEK and ERK activity, ERK dependent phosphorylation of MEK and cytokine gene expression. Similar experiments will investigate the function of CL100 and PAC1 phosphatases in vivo in mediating the deactivation of ERK in T-cells and determine the consequences of this ERK deactivation mechanism on T-cell signal transduction. This will involve similar transient transfection experiments but including overexpression of CL100 and PAC1 phosphatases.